Loop diuretics are widely used to treat premature infants in neonatal intensive care units. Recent reports have suggested an increased risk of permanent sensorineural hearing loss among infants receiving such treatments. Recent studies of an animal model carried out by the principal investigator have demonstrated increased ototoxicity of furosemide at certain stages of development. The basis for the increased susceptibility to furosemide ototoxicity during neonatal development appears to be multifactorial. Several factors which undergo ontogenetic change and which could influence ototoxic sensitivity to loop diuretics include; glucose utilization/energy metabolism in the cochlea (Aim 1) drug distribution and an immature blood-labyrinth barrier (Aim 2) hepatic metabolism of xenobiotics (Aim 3) and renal excretion of drugs (Aim 4). This research program seeks to uncover the determinants which underlie the increased susceptibility to furosemide ototoxicity in the neonatal rat as a model for human neonatal furosemide ototoxicity. Aim 1 will investigate the effect of furosemide in the cochlea on the activities of key glycolytic enzymes, hexokinase (HK) 6-phosphofructo-1-kinase (PKE) and pyruvate kinase (PK). Preliminary experiments have demonstrated that furosemide is a potent noncompetitive inhibitor of PFK. The brain enzyme was more strongly inhibited than the liver or muscle isozymes. If the inhibitory effects of furosemide on PFK and glucose utilization are a cause of furosemide ototoxicity, then relief of the inhibition of PFK should at least partially ameliorate ototoxicity. Since probenecid both increases hyperglycemia and reduces ototoxicity due to furosemide, it is possible that increased availability of glucose promotes increased glucose utilization and consequently improved energy metabolism by the cochlea. This effect could be mediated by increased levels of fructose-2,6-bisphosphate in the cochlea. Another aspect of Aim 1 is to pharmacologically manipulate the levels of fructose-2,6-bisphosphate to relieve furosemide ototoxicity. The effects of glucagon-induced hyperglycemia and insulin-induced hypoglycemia on furosemide ototoxicity will be investigated. The second specific aim will be to test the hypothesis that increased susceptibility of the developing cochlea to furosemide is mediated in part by pharmacokinetic factors. Furosemide and furosemide-glucuronide will be measured in plasma, perilymph, cochlear tissues and urine with liquid chromatography (HPLC). The third specific aim will be to examine the influence of drug metabolism on ototoxicity in development. Hepatic UDP-glucuronyl transferase (UDP-GT) will be induced by phenobarbital to determine whether this reduces furosemide ototoxicity by accelerating its metabolism and excretion. The fourth aim is to examine the role of immature renal excretion on the enhanced susceptibility to loop diuretic ototoxicity. Renal tubular secretion will be induced by substrate stimulation using penicillin pretreatment. This should enhance tubular secretion which is the primary route for furosemide excretion./ These studies should provide a much greater understanding of the effect of various factors which may play key roles in the production of enhanced susceptibility to furosemide ototoxicity in immature neonates. Further these findings could provide insight into multiple therapeutic approaches to ameliorate ototoxicity and help to optimize the desired effects of loop diuretics in neonates.